This proposal aims to develop the new capability of using single molecule fluorescence (or Forster) resonance energy transfer (sm FRET) from proteins within living cells to observe real time conformational dynamics in parallel with spatial localization of individual proteins as they circulate within cellular networks. Experiments that follow single molecules can uncover properties that are impossible to observe in bulk measurements due to the inherent averaging over molecules and over time, and lack of synchronization. Single particle measurements of dynamic conformations of individual molecules can determine multiple reaction pathways and transient intermediate states as well as exact distributions of molecular properties. The general approach in this proposal is to use high resolution structural information already available for the proteins under study to design site-specific labeling mutants. These mutants will be produced, purified and labeled free of cells and then microinjected into cultured eukaryotic cells. Fluorescence microscopy and spectroscopy will be used to track individual protein molecules as well as determine the degree of FRET from that molecule in real time as they circulate within living cells. This untested approach, if successful, will have broad applicability to many biological questions and has the potential to reveal details of cellular networks that are not observable by any other technique. This approach will be applied within 2 different physiological networks to determine, 1) the spatial distribution and detailed sequence of folding/unfolding transitions in SNARE proteins involved in intracellular vesicle transport and membrane fusion, and 2) the activation of Protein Kinase A in second messenger signaling. Advances in understanding of cellular regulatory and signaling networks will have broad impact in all areas of health related research: understanding of the disease state, homeostasis and development. Ultimately knowledge derived with these methods will improve human health. [unreadable] [unreadable] [unreadable]